This proposal is a renewal application of the parent R01 project (AI095066). This application builds upon our pioneering accomplishments made during the parent period wherein we successfully demonstrated that genomic signatures within the intrahost HIV sequence population are precise and robust markers to distinguish incident from chronic infections. These genomic biomarkers, measuring the presence of closely related strains as a signature of incidence, showed over 95% accuracy. We further refined the incidence assay by implementing cutting-edge technology to design a high-throughput, next-generation sequencing platform. This assay is currently being actively tested on over 600 incident and chronic specimens in collaboration with preeminent centers with well-established cohorts. In the present application, we propose to develop a highly accurate and low cost genomic assay which concurrently determines HIV incidence and profiles transmitted drug resistance mutations (TDRMs) from a single measure in cross-sectional surveys. Major innovations of the proposal include i) integrating a high-throughput next-generation sequencing platform with a bioinformatics pipeline, statistical tests and mathematical modeling, ii) designing novel statistical methods to objectively determine the False Recency Rate (FRR) and mean duration of recent infection (MDRI) - the standard incidence assay evaluation criteria - and iii) distributing easily accessible web-based software with which the global HIV community can interact for incidence and TDRM surveillance. Our genomic assay will first produce next-generation sequencing reads of envelope gene segments to report the stage of infection. Using our new statistical hierarchical Bayesian models, we will analyze the genomic biomarker dynamics over time to calculate the MDRI and FRR. We will then monitor the presence of 82 WHO-listed commonly occurring TDRMs associated with the Department of Health and Human Services' recommended and alternative therapy regimens. Our single assay approach responds to the growing need to precisely identify TDRMs as their global presence increases. The validity of the proposed assay will be extensively tested with a large volume of HIV sequences from diverse populations, including i) well-established cohorts from preeminent centers (WIHS, CDC, CHAVI, and CEPHIA), ii) our own cohorts at the Rand Schrader Clinic at USC and Los Angeles Gay and Lesbian center, and iii) the virologic failure cohort (A5202) from the Clinical AIDS Trial Group. This project's end-product, web-based software, will systematically produce and organize vital information to inform optimal therapy regimens for individuals and geographically trace population-wide incidence and TDRM trends. The proposed work will advance HIV prevention efforts by providing a working incidence and TDRM surveillance assay ready for direct use in cross-sectional epidemic monitoring.